Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0608—Germ cells
  • C12N5/0612—Germ cells sorting of gametes, e.g. according to sex or motility
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
  • G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0652—Sorting or classification of particles or molecules
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0627—Sensor or part of a sensor is integrated
  • B01L2300/0645—Electrodes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
  • C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/04—Cell isolation or sorting

Definitions

• the present invention relates to the field of in vitro discrimination of spermatozoa according to their X or Y chromosome, especially spermatozoa, labeled with at least one fluorochrome.
• a spermatozoon is a haploid type cell, which usually contains only one copy of each chromosome, for example an X or Y chromosome, and whose motility is ensured by a flagellum.
• sperm is labeled with a fluorescent DNA spacer, generally referred to as a fluorochrome;
• X spermatozoa which are presumed to contain a greater amount of DNA, absorb a larger amount of fluorochrome than Y spermatozoa;
• each X or Y sperm is automatically sorted according to this detected fluorescence intensity.
• a first known sorting method (step (e) above) consists in subjecting the solution containing the marked spermatozoa to vibrations by means of a piezoelectric transducer, so as to form microdroplets, which as far as possible each contain only a single sperm, then electrically charging each droplet according to the detected fluorescent intensity, and finally passing successively the electrically charged microdroplets in an electric field to separate the charged droplets containing an X sperm from the charged droplets containing a spermatozoon. sperm Y.
• This sorting method is described for example in the following international patent applications: WO2004 / 104178, WO2004 / 017041, WO2009 / 014643, WO2009 / 151624.
• the object of the present invention is to propose a novel technical solution for discriminating spermatozoa labeled with at least one fluorochrome, as a function of their X or Y chromosome, which notably overcomes the aforementioned drawbacks of the solutions of the prior art.
• the subject of the invention is a device for discriminating labeled spermatozoa by means of at least one fluorochrome, which device comprises a transport channel, in which a solution containing said labeled spermatozoa can be circulated in a direction of predefined transport (Y), excitation means for exciting the fluorescence emission of labeled spermatozoa circulating in said transport channel, means for detecting the fluorescence emitted by the marked spermatozoa circulating in said transport channel, and means electronic treatment for discriminating labeled spermatozoa circulating in the transport channel from the fluorescence detected by the fluorescence detection means:
• said transport channel comprises a principal plane of symmetry (P1) parallel to the direction of transport ( Y), and a cross section, in a plane (X, Z) perpendicular to the conveying direction (Y), which is symmetrical, which is characterized by a main axis of symmetry (A) and has a maximum transverse dimension (H) ), measured along this principal
• the invention also relates to a device for discriminating labeled spermatozoa by means of at least one fluorochrome, which device comprises a transport channel, in which a solution containing said labeled spermatozoa can be circulated in one direction.
• predefined transport device Y
• excitation means for exciting fluorescence emission of labeled spermatozoa circulating in said transport channel
• electronic means of treatment for discriminating spermatozoa labeled circulating in the transport channel from the fluorescence detected by the fluorescence detection means
• said device comprises means for transporting the spermatozoa one behind the other in the transport channel with substantially the same spatial orientation of each spermatozoon
• the fluorescence detection means comprise at least first means of which are oriented in the direction of one of the two main faces of larger area of each spermatozoid passing successively in the transport channel in front of said detection means being oriented spatially
• the electronic processing means are capable of automatically discriminating the spermatozoa by using at least the duration of each fluorescence signature characteristic of a spermatozoon in the fluorescence detection signal delivered by the first detection means.
• the device of the invention may comprise the following additional and optional features, taken alone or in combination with each other and defined in any one of claims 2 to 15 or 17 to 30.
• the subject of the invention is also a use of the abovementioned device for automatically discriminating, and where appropriate automatically selecting or sorting automatically, in vitro spermatozoa.
• the subject of the invention is also a process for the in vitro discrimination of spermatozoa during which a solution containing the spermatozoa labeled with at least one fluorochrome is injected into the transport channel of the above-mentioned device and circulated in the canal. transporting said spermatozoa one behind the other spatially orienting them with the same spatial orientation, and automatically detecting the chromosome type of each spermatozoon circulating in the transport channel, using at least the duration (D) of the signature of characteristic fluorescence of a spermatozoon in the fluorescence detection signal delivered by the first detection means of the device.
• D duration
• the subject of the invention is also a method for producing a sexed semen by in vitro screening or sorting of spermatozoa by means of the above-mentioned device, during which a solution containing the spermatozoa labeled with the medium is injected into the transport channel of the device.
• At least one fluorochrome is circulated in the transport channel said spermatozoa one behind the other spatially orientated with the same spatial orientation, it automatically detects the type of chromosome of each sperm circulating in the transport channel, and especially if the spermatozoon is of type X or Y, using at least the duration (D) of each fluorescence signature characteristic of a spermatozoon in the fluorescence detection signal delivered by the first detection means of the device, and selecting spermatozoa of a given type, in particular by altering the spermatozoa of the other type, or spermatozoa according to their type, and in particular of their type X or Y, in particular separating the spermatozoa of a given type from the spermatozoa of the other type.
• D duration
• the subject of the invention is also sexed semen, and in particular semen of mammals, and more particularly a sexed semen of cattle, pigs, sheep, equines, goats, or rabbits, obtained by using the above-mentioned device. by implementing the aforementioned production process.
• FIG. 1 is a schematic representation of a first embodiment of a selection device of the invention, the microfluidic chip of this selection device being shown in plan view;
• FIG. 2 is a cross-sectional view of the microfluidic chip of this selection device, in the sectional plane II-II of FIG. 1;
• FIG. 3 is a cross-sectional view of this sorting device, in the section plane III-III of FIG. 1;
• FIG. 4 is a cross-sectional view of the sorting device in the section plane IV-IV of Figure 3;
• FIG. 5 is a cross-sectional view of this sorting device, in the sectional plane V-V of FIG. 1;
• FIG. 6 is a cross-sectional view of this sorting device, in the section plane VI-VI of FIG. 1;
• FIG. 7 is a cross-sectional view of this sorting device, in the section plane VII-VII of FIG. 1;
• FIG. 8 is a cross-sectional view of this sorting device, in the section plane VIII-VIII of FIG. 7;
• FIG. 9 is a cross-sectional view of a second variant of a selection device
• FIG. 10 is a cross-sectional view of the transport channel on which two fluorescence detection cones C1 and C2 have been schematized;
• FIG. 11 represents two exemplary chronograms of two fluorescence detection signals of a device of the invention.
• FIG. 12 schematically represents an example of a fluorescence signature (PIC) in a fluorescence detection signal
• FIG. 13 is an example of a histogram of the maximum of the fluorescence intensity detected at the bottom of the transport channel in a device of the type of that of FIG. 1;
• FIG. 14 is an example of a histogram of the durations (D) of the fluorescence signatures detected laterally in a device of the type of that of FIG. 1;
• FIG. 15 is an example of a histogram of the durations (D) of the fluorescence signatures detected at the bottom of the transport channel in a device of the type of that of FIG. 1;
• FIG. 16 represents three examples of chronograms respectively of the two fluorescence detection signals and of the signal for controlling the selection means of the device of FIG. 1 which is generated automatically from these two fluorescence detection signals.
• FIG. 1 shows a first embodiment of a device for discriminating and selecting spermatozoa according to their X or Y chromosome.
• This device of the invention can be used to select in vitro X or Y spermatozoa, and more particularly can be used in the field of animal reproduction to select in vitro any type of X or Y spermatozoa, and in a nonlimiting manner and not exhaustive, spermatozoa of cattle, pigs, sheep, equines, goats, rabbits.
• This device comprises:
• microfluidic chip 1 comprising a main microfluidic transport channel 100
• injection means 2 for injecting, into the main microfluidic transport channel 100, a solution S containing a sample of spermatozoa SP to be discriminated and selected, hydrodynamic focusing means (3, 101) for injecting, into the main microfluidic transport channel 100, a liquid L (compression fluid) so as to obtain a hydrodynamic focusing of the spermatozoa in the main microfluidic transport channel 100 ,
• selection means 6 comprising a source of electromagnetic radiation 60, and which makes it possible to selectively alter the spermatozoa circulating in the main microfluidic transport channel 100, by means of the electromagnetic radiation emitted by the source 60,
• collection means 8 making it possible to collect all the spermatozoa at the output of the main microfluidic transport channel 100.
• the microfluidic chip 1 comprises a rigid substrate 10, having a front face 10a and a rear face 10b, and a plate 11 attached to the front face 10a of the substrate 10.
• the main microfluidic transport channel 100 is rectilinear and extends in the longitudinal direction Y corresponding to the direction of movement of the spermatozoa in the microfluidic channel 100.
• This microfluidic channel 100 completely crosses the substrate 10 and has an inlet opening 100a and a discharge opening 100b.
• all or part of this main microfluidic channel 100 may not be rectilinear.
• the cross-section (in a plane (X, Z)) of the main microfluidic transport channel 100 is rectangular, of height H measured in the direction Z perpendicular to the flat upper face 10a of the substrate 10, and width E measured in the transverse direction X, parallel to the upper face 10a of the substrate 10 and perpendicular to the longitudinal direction Y of the microfluidic channel.
• the height H of the channel 100 is greater than the width E of the microfluidic channel OO.
• the width E of the channel is less than 1 mm, and more preferably still less than ⁇ ⁇ .
• the height H of the channel may also be less than 1 mm.
• the main microfluidic channel 100 is delimited by a groove having a U-shape in cross section and made in the upper face 10a of the substrate 10, and by the face rear 1 1 b of the plate 1 1.
• the bottom wall 100c of the groove forms the bottom wall of the microfluidic channel 100; the two walls 100d of the groove which are parallel, and which are transverse, and more particularly perpendicular to the bottom wall 100c, form the two longitudinal walls 100d of the microfluidic channel 100; the part of the lower face 1 1 b of the plate 1 1, located in line with the U-shaped groove, forms the upper wall 100e of the microfluidic channel 100.
• This rectangular cross section of the microfluidic channel 100 makes it possible to contribute to the spatial orientation of the spermatozoa in the channel 100, in particular in combination with the hydrodynamic focusing.
• the spermatozoa having a non-spherical and flattened shape the implementation of a microfluidic channel 100 with a rectangular cross-section contributes to a spatial orientation of the spermatozoa with their flattened faces F of larger surface oriented substantially parallel to the plane (Y , Z), that is to say substantially parallel to the plane of the two longitudinal walls 100d of the microfluidic channel 100. It is up to one skilled in the art to select judiciously and in a manner known per se the dimensions of the microfluidic channel 100 , and in particular the ratio H / E.
• transport channel 100 having a rectangular cross section
• said transport channel 100 may more generally have a different geometry in cross section contributing to the above-mentioned spatial orientation of spermatozoa.
• a transport channel 100 having a main plane of symmetry P1 parallel to the transport direction Y, and comprising, in a plane X, Z perpendicular to the direction of transport, a cross section, which is symmetrical, which is characterized by a principal axis of symmetry A ( Figure 2), and which has a maximum transverse dimension H, measured along this main axis of symmetry A, and a minimum transverse dimension E, less than the transverse dimension H, and measured along a secondary transverse axis B ( Figure 2) perpendicular to the main axis of symmetry A.
• the transport channel 100 may have in cross section a oval, elliptical shape, ...
• the substrate 10 is made of a material, which is chemically inert.
• the material of the substrate 10 is chosen so as to be able to undergo a physical or chemical etching, for example a plasma etching. More particularly, but not limited to the invention, the substrate is for example silicon or gallium arsenide.
• the groove (100c, 100d) of height H and width E is advantageously achieved by anisotropic etching of the upper face 10a of the substrate 10.
• the substrate with the groove (100c, 100d) can also be produced by any another known manufacturing process and for example by 3D printing or by a so-called hot embossing technology.
• the substrate may also be made of a polymeric material.
• Plate 11 is in a material that is chemically inert, and may be opaque or transparent.
• the plate 1 1 is for example glass or plastic. It is fixed to the substrate 10 by any means, and for example by anodic bonding or thermocompression.
• the injection means 2 have the function of injecting, into the main microfluidic channel 100, a solution S containing a sample of spermatozoa to be selected.
• these injection means 2 comprise a syringe 20, which is filled with a solution S containing the specimen of spermatozoa SP to be selected, and which is associated with an automatic injection system. 21, which may for example be of the syringe pump type or peristaltic pump type.
• the outlet of the syringe 20 is coupled to a capillary tube 22 whose distal portion 22a has been inserted into the microfluidic channel 100 through the inlet opening 100a of this channel 100.
• the capillary tube 22 is a flexible or rigid tube , whose cross section is preferably adapted to the section of the microfluidic channel 100.
• the capillary tube 22 is preferably attached to the substrate 10, by any means, and in particular by gluing.
• SP spermatozoa are diluted in buffer solution S, which is biologically compatible with spermatozoa, and for example in an aqueous solution containing 30 g / L of TRIS (trishydroxymethylaminomethane), 17.25 g / L of citric acid monohydrate and 12, 5 g / L fructose in water at a pH of about 7.
• buffer solution S which is biologically compatible with spermatozoa
• TRIS trishydroxymethylaminomethane
• citric acid monohydrate 17.25 g / L of citric acid monohydrate and 12, 5 g / L fructose in water at a pH of about 7.
• the SP spermatozoa DNA contained in the buffer solution S has been labeled, in a manner known per se, by means of at least one fluorochrome, which can fluoresce when it is associated with the DNA.
• fluorochromes commonly used to mark spermatozoa, mention may be made, by way of non-limiting and non-exhaustive examples: bischenzimide-type fluorochromes, and in particular Hoechst fluorochromes (Hoechst 33342, Hoechst 33258, ...), bromide d ethidium, SYBR fluorochromes such as SYBR-14.
• the injection system 21 pushes the buffer solution S containing the SP spermatozoa, so as to get it out through the distal opening 22b of the capillary 22, and to inject it into the microfluidic channel 100 with a controlled flow automatically and preferably constant.
• a controlled flow automatically and preferably constant.
• the microfluidic chip 1 comprises two secondary microfluidic channels 101, which in the usual way are formed on either side of the microfluidic transport channel 100 ( Figure 1 ).
• Each secondary microfluidic channel 101 has an inlet opening 101a and opens, opposite the inlet opening 101a, laterally in the main microfluidic channel 100.
• the outlet of the capillary tube 22 is positioned upstream of the junction zone between the secondary microfluidic channels 101 and the microfluidic transport channel 100, the distal portion 22a of the capillary tube 22 being inserted more or less deeply into the transport channel 100 .
• each secondary microfluidic channel 101 is delimited on the one hand by a U-groove etched in the upper face of the substrate 10, and on the other part by the lower face 1 1 b of the plate 1 1.
• the hydrodynamic focusing means comprise, for each secondary channel 101, injection means 3 in the form of a syringe 30, which is filled with a solution L, and which is associated with an automatic injection system 31, for example type syringe pump or peristaltic pump type or any other means for actuating liquid.
• the outlet of the syringe 30 is coupled to a capillary tube 32, the distal portion 32a of which has been inserted into the microfluidic channel 100 through the inlet opening 101a of a secondary channel 101.
• Each capillary tube 32 is a flexible tube, the cross section of which is preferably adapted to the section of the secondary channel 101. More in particular, each capillary tube 32 is preferably fixed to the substrate 10, by any means, and in particular by gluing.
• the liquid used for the L solutions is preferably, but not necessarily, identical to that used for the S buffer solution containing SP spermatozoa.
• each injection system 31 pushes each solution L so as to inject it into the corresponding secondary microfluidic channel 101 with an automatically controlled, and preferably constant, flow rate.
• each solution L and the buffer solution S containing SP spermatozoa are automatically controlled, so as to create in the microfluidic channel 100a, two high speed laminar flow FL formed by each solution L, of either side of the slower central flow formed by the solution S containing SP spermatozoa.
• These laminar flow FL allow, in known manner, SP sperm to be trained in the main microfluidic channel 100 by subjecting them to a hydrodynamic focusing, of the 2D type, which substantially results in aligning the spermatozoa SP one behind the other, with a SP spermatozoa alignment substantially in a longitudinal hydrodynamic focusing plane parallel to the main plane of symmetry P1 of the transport channel 100.
• the rectangular section or equivalent of the transport channel 100 provides a spatial orientation of the sperm with their flattened face a larger surface oriented substantially parallel to the main plane of symmetry P1 of the transport channel 100, that is to say substantially parallel to the plane of the two longitudinal walls 100d of the microfluidic channel 100 in the variant of Figures 1 and 2.
• the position of this hydrodynamic focusing plane of the spermatozoa between the two longitudinal walls 100d of the channel 100 depends in particular on the flow difference between the two laminar flows.
• the plane of focus hydrodynamic spermatozoa is substantially centered between the two longitudinal walls 100d of the channel 100 ( Figure 2), that is to say substantially corresponds to the main plane of symmetry P1 of the transport channel 100.
• the The hydrodynamic focal plane of the spermatozoa may nevertheless be off-center between the two longitudinal walls 100d of the channel 100 with respect to the main plane of symmetry P1.
• the invention is not limited to a 2D hydrodynamic focusing of spermatozoa SP in the main microfluidic channel 100. It is also possible in the context of the invention to implement a 3D hydrodynamic focusing, as described for example in the International Patent Application WO201 1/005776, so as to align the spermatozoa substantially along a longitudinal hydrodynamic focusing axis parallel to the Y axis of the microfluidic channel 100.
• the aforementioned particular injection means 3 can be replaced by any equivalent injection system making it possible to inject, in the main microfluidic channel 100, with a controlled flow, the solutions L to obtain, in a manner known per se, said 2D hydrodynamic focusing or 3D spermatozoa in the transport channel 100.
• the fluorescence excitation means 4 comprise a source 40 of electromagnetic radiation, of the laser source type, the wavelength of which is adapted to the marker (fluorochrome) of the spermatozoa.
• electromagnetic excitation fluorescence excitation of wavelength of between 300 nm and 400 nm is used, for example more particularly at around 375 nm when SP spermatozoa have been marked with Hoechst.
• the microfluidic chip 1 comprises a first through passage
• this through passage 102 is formed through one of the longitudinal walls 100d of the microfluidic channel 100.
• the distance between the outlet 102b in the transport channel 100 of the through passage 102 and the opposite wall 100d of the transport channel 100 corresponds to the width E of the transport channel 100, and is preferably less than 1 mm, more preferably less than ⁇ ⁇ .
• This through passage 102 is delimited by a U-groove etched in the upper face 10a of the substrate 10 and by the lower face 11b of the plate 11.
• the through passage 101 could be pierced through the substrate 10.
• the output of the electromagnetic radiation source 40 is coupled to an optical fiber 41 whose distal portion 41a is inserted into this first through passage 102, so that the transmission end 41b ( Figure 4) of the fiber optical 41 does not project in the microfluidic channel 100, so as not to disturb the flow of fluids in the microfluidic channel 100, and thus not to disturb the positioning and spatial orientation of SP spermatozoa circulating in the channel Microfluidic 100.
• the emission end 41b of the optical fiber 41 is positioned closer to this microfluidic channel 100, and preferably flush with the microfluidic channel 100.
• the mechanical cladding of the optical fiber is referenced 412
• the optical cladding of the optical fiber is referenced 410
• the core of the optical fiber is referenced 41 1.
• the distal end of the optical cladding 410 and the core 41 1 of the optical fiber, through which electromagnetic excitation radiation is emitted, is stripped.
• the distal end of the optical cladding 410 and the core 41 1 of the optical fiber could not be stripped and be surrounded by the mechanical sheath 412 of the optical fiber 41.
• the through passage 102 is profiled to include a shoulder 102a forming a positioning stop 102a for the distal end 412a of the mechanical sheath 412 of the optical fiber. It suffices to insert the optical fiber until the distal end 412a of the mechanical sheath 412 is blocked by the positioning stop 102a, which advantageously allows a simple and precise positioning of the distal end of emission of the optical fiber 41 with respect to the microfluidic channel 100.
• the electromagnetic excitation radiation is emitted by the optical fiber 41 directly into the transport channel 100.
• the transmission end of the optical fiber 41 is positioned slightly recessed in the through passage 102, the electromagnetic excitation radiation is emitted in this through passage 102 and then enters the transport channel 100.
• This insertion of the optical fiber 41 into the microfluidic chip 1, close to the microfluidic channel 100, advantageously makes it possible to bring the electromagnetic excitation radiation as close as possible to the spermatozoa SP circulating in the microfluidic channel, which contributes to improving the performances. the excitation of the fluorescence.
• the distance Di (FIG. 4), between the output 102b in the transport channel 100 of the first through-passage 102 and the hydrodynamic focusing plane (2D hydrodynamic focusing) or the hydrodynamic focusing axis (3D hydrodynamic focusing) of the spermatozoa SP in the transport channel 100 is preferably very small.
• the path length of the electromagnetic excitation radiation is reduced to the spermatozoa SP, through the sperm transporting liquid in the transport channel 100.
• this distance Di is less than 1 mm, more preferably less than ⁇ ⁇ , and more preferably still less than 50 ⁇ .
• the insertion of the optical fiber 41 into the microfluidic chip 1 also makes it possible to avoid the risks of misalignment of the electromagnetic excitation radiation with respect to the microfluidic channel 100 when handling the microfluidic chip 1.
• the invention is however not limited to the implementation of an optical fiber 41 integrated in the chip and opening into the transport channel 100.
• the means 4 for excitation of the fluorescence may comprise a light excitation source (not necessarily fiberized) which is external to the microfluidic chip 1 and which allows illumination of the spermatozoa by means of excitation radiation emitted outside the microfluidic chip and passing through a wall of the transport.
• the fluorescence detection means 5 comprise a first optical fiber 51 and a photo-detector 50, of the photomultiplier (PM) type, which is adapted to detect the fluorescence wavelength of the spermatozoa, namely for example a length of wave between 400nm and 500nm, and for example around 460nm when the sperm were marked with a fluorochrome Hoechst type.
• PM photomultiplier
• the microfluidic chip 1 comprises a second through passage 103, which opens into the main microfluidic channel 100 (FIGS.
• this through passage 103 is formed through the other longitudinal wall 100d of the microfluidic channel 100.
• the distance between the output 103b in the transport channel 100 of the through passage 103 and the opposite wall 10Od of the transport channel 100 corresponds to the width E of the transport channel 100, and is preferably less than 1 mm, more preferably less than ⁇ ⁇ .
• the mechanical cladding of the optical fiber is referenced 512
• the optical cladding of the optical fiber is referenced 510
• the core of the optical fiber is referenced 51 1.
• the distal portion 51a of the detection optical fiber 51 is inserted into the second through-passage 103 so that the distal end of the optical fiber 51 not protruding into the microfluidic channel 100, and is preferably positioned closer to this microfluidic channel 100.
• This optical fiber 51 makes it possible to detect the fluorescence emitted by the spermatozoa in a direction substantially perpendicular to the main plane of symmetry P1 of the transport channel 100.
• the fluorescence detection means detect at least a portion of the fluorescence emitted by the SP spermatozoa inside at least one detection cone of at most 90 °, with an axis perpendicular to the main plane of symmetry P1 and whose vertex is oriented towards the transport channel 100 (FIG. 0 / cone C1 with an angle at the apex of 90 °).
• these means for detecting fluorescence can detect the fluorescence emitted by SP spermatozoa at least in a direction perpendicular to the main plane of symmetry P1.
• the first fluorescence detection means and in this case in this particular embodiment the distal end the first optical fiber 51, are oriented in the direction one of the two main faces F of greater area of each SP spermatozoid passing successively in the transport channel in front of said detection means.
• the transmitting end 51b of the detection fiber 51 is positioned opposite the photodetector 50, so that the light (fluorescence) which is emitted in the microfluidic channel 100, and which is picked up by the fiber 51, is detected by the light detector 50.
• the light detector 50 delivers an electrical signal 50a characteristic of the luminous intensity of the fluorescence which is detected.
• the distance between the output 103b in the transport channel 100 of the second through-passage 103 (FIG. 1 - opening 103b of this passage 103 opening into the transport channel 100) and the hydrodynamic focusing plane P (2D hydrodynamic focusing) or the hydrodynamic focusing axis (3D hydrodynamic focusing) of the spermatozoa in the transport channel 100 is preferably very small.
• the optical detection fiber 51 may for example be a broad-core optical fiber 51 1 (FIG. 5) in contrast to the excitation optical fiber 41, whose core 41 1 (FIG. ) is of smaller diameter so as to spatially focus the electromagnetic radiation.
• the fluorescence detection means 5 also comprise a second optical fiber 52 associated with a photo-detector 53, of the photomultiplier (PM) type, which is adapted to detect the fluorescence wavelength of the spermatozoa, that is to say for example a wavelength between 400nm and 500nm, and for example around 460nm when the spermatozoa were marked with a fluorochrome Hoechst type.
• PM photomultiplier
• the microfluidic chip 1 comprises a third through passage 104, which is formed through the bottom wall 100c of the microfluidic channel 100 and which opens into the main microfluidic channel 100 in the same zone as the optical fibers 41. and 51 above.
• the mechanical sheath of the optical fiber is referenced 522
• the optical cladding of the optical fiber is referenced 520
• the core of the optical fiber is referenced 521.
• the distal portion 52a of the detection optical fiber 52 is inserted into this third through passage 104 so that the distal end of the optical fiber 52 is not not protruding into the microfluidic channel 100, and is preferably positioned closer to this microfluidic channel 100.
• the transmitting end 52b (FIG. 1) of the detection fiber 52 is positioned opposite the photodetector 53, so that the light (fluorescence) which is emitted in the microfluidic channel 100, and which is captured by the fiber 52 at the bottom of the transport channel, is detected by the light detector 53.
• the light detector 53 delivers a signal Electrical 53a ( Figure 1) characteristic of the fluorescence light intensity that is detected.
• the detection optical fiber 52 may for example be a wide-core optical fiber 521 (FIG. 6).
• this second detection optical fiber 52 makes it possible to detect the fluorescence emitted by the spermatozoa in a direction that is essentially perpendicular and more particularly perpendicular to the secondary plane P2 of the transport channel that is perpendicular to the main plane of symmetry P1.
• the means for detecting fluorescence and in this case in this particular variant embodiment, the first optical fiber 52, detect at least a part of the fluorescence emitted by the spermatozoa SP at least within a detection cone of at most 90 °, with an axis perpendicular to the secondary plane P2, and whose apex is directed towards the transport channel 100 (figure O / cone C2 angle ⁇ at the apex of 90 °).
• these fluorescence detection means make it possible to detect the fluorescence emitted by SP spermatozoa at least in a direction perpendicular to the secondary plane P2.
• the second means for detecting fluorescence and in this particular embodiment in this particular embodiment the distal end of the first optical fiber 52, are oriented in direction of one of the two slices T of smaller thickness SP sperm successively in the transport channel 100 in front of said detection means.
• the invention is not limited to the use of optical fibers associated with photo-detectors, of the photomultiplier (PM) type, to detect the fluorescence emitted by the spermatozoa, but the optical fibers 51 and 52 and the photomultipliers. detectors can be replaced by any other means of fluorescence detection.
• PM photomultiplier
• the selection means 6 comprise a source 60 of electromagnetic radiation, for example of the laser source type (pulsed or continuous) and an optical fiber 61.
• the output of this source of electromagnetic radiation 61 is coupled to the optical fiber 61.
• the microfluidic chip 1 comprises a fourth through passage 105, which opens into the main microfluidic channel 100 (FIGS. 1 and 7) in a portion of the main microfluidic channel 100 located downstream of the fluorescence detection zone.
• this through passage 105 is made through one of the longitudinal walls 100d of the microfluidic channel 100.
• the distance between the outlet 105b in the transport channel 100 of the through passage 105 and the opposite wall 100d of the transport channel 100 corresponds to the width E of the transport channel 100, and is preferably less than 1 mm, more preferably less than ⁇ ⁇ .
• the optical cladding of the optical fiber is referenced 610, and the core of the optical fiber is referenced 61 1.
• the distal portion 61a of the optical fiber 61 is inserted into this fourth through-passage 105, so that the distal emitting end 61b (FIG. 7) of the optical fiber 61 does not protrude into the microfluidic channel 100, and is preferably positioned closer to this microfluidic channel 100.
• the source 60 When activated, the source 60 emits in the microfluidic channel 100 an electromagnetic radiation of alteration R, whose function is to directly or indirectly alter an SP spermatozoon circulating in the main microfluidic channel 100 and passing through this electromagnetic radiation. alteration, so that this spermatozoon is no longer fertile.
• the spermatozoon SP passing through the electromagnetic radiation of alteration is modified, so that its viability or motility is sufficiently deteriorated so that the spermatozoid is no longer fertile, regardless of the physical and / or biological and / or chemical phenomenon causing this deterioration.
• the wavelength of the altering electromagnetic radiation will typically be selected in a range of wavelengths from UV to IR.
• the electromagnetic radiation of alteration is emitted by the optical fiber 61 directly into the transport channel 100.
• the electromagnetic radiation alteration is emitted in this through passage 105 and then enters the transport channel 100.
• the distal portion 61a of the optical fiber 61 into the microfluidic chip, the risks of misalignment of the electromagnetic radiation of alteration with respect to the microfluidic channel 100, especially when handling the microfluidic chip 1.
• the invention is however not limited to the implementation of an optical fiber 61 integrated in the chip and opening into the transport channel 100.
• the selection means may for example comprise a source of radiation which provides illumination for the selection of sperm by means of radiation emitted outside the microfluidic chip and passing through a wall of the transport channel.
• the previously described fluorescence excitation means 4 may be replaced by fluorescence excitation means 4 'which comprise a source 40' of electromagnetic radiation, for example laser source type, and which are adapted to emit electromagnetic radiation excitation REF, the wavelength of which is adapted to the marker (fluorochrome) spermatozoa.
• This electromagnetic excitation radiation REF is emitted outside the microfluidic chip, and passes through the upper wall 1 1 of the transport channel in the direction of SP spermatozoa circulating in this transport channel.
• each spermatozoon SP When SP spermatozoa circulate in the transport channel 100, due in particular to the cross-sectional geometry of the transport channel, each spermatozoon SP is spatially oriented so that its main surface F of larger area is substantially parallel to the plane of symmetry. main P1 of the transport channel.
• spermatozoa unlike the aforementioned variant implementing lateral lighting (fluorescence excitation means 4 referred to above).
• the fluorescence excitation means 4 ' may also be adapted to emit electromagnetic excitation radiation R outside the microfluidic chip, and through the bottom wall 10 of the transport channel.
• this lighting could also be carried out directly in the transport channel by means of an optical fiber, which is integrated in the microfluidic chip by being passed through the upper wall 11 or the bottom wall 10 of the transport channel.
• an electromagnetic excitation radiation REF propagating at least in a direction substantially parallel to the main plane of symmetry P1 of the transport channel.
• the electronic processing and control means 7 receive at input the two fluorescence detection signals 50a, 53a respectively delivered by the photo-detectors 50 and 53, and output a control signal 7a for automatically controlling said source of radiation. electromagnetic 60, from the fluorescence detection.
• Fluorescence detection assays were conducted with Hoechst 33342-labeled sexed cattle semen and passed through a microfluidic device of the invention.
• the following considerations and the invention are not limited to the semen of cattle, but can be generalized to the semen of any type of mammal comprising X or Y chromosomes, and in particular to sperm of pigs, sheep, equidae, goats, rabbits.
• FIG. 11 shows two examples of fluorescence detection time signals 50a (lateral detection) and 53a
• bottom channel detection when a mammalian spermatozoon SP, in the occurrence of a bovine, passes in the transport channel in front of the distal ends of the two optical fibers 51 and 52.
• FIG. 12 also shows a schematic example of a fluorescence detection time signal 50a or 53a when a mammalian spermatozoon SP, in this case a bovine, passes in the transport channel in front of the distal end of the optical fiber 51 or 52.
• the passage of a spermatozoon in front of the distal end of the optical fiber 51 or 52 results in a temporal variation in the intensity of the fluorescence detection signal 50a or 53a.
• This temporal variation of the intensity of the fluorescence detection signal 50a or 53a forms, in the fluorescence detection signal 50a or 53a, a signature which is referenced PIC in the figures, and during which the intensity of the detection signal is constantly greater than a minimum fluorescence threshold Smin predefined, and preferably parameterizable.
• this PIC signature above said predetermined minimum threshold Smin of fluorescence and characteristic of the passage of a spermatozoon in front of the distal end of the optical fiber 51 or 52 has been indicated.
• This minimum threshold Smin is set by those skilled in the art so as to filter the noise in the detection signal 50a or 53a and to allow a detection of the PIC fluorescence signature by the electronic processing means 7 and a measurement of the duration D of this PIC signature during which the fluorescence intensity is above this threshold Smin.
• the maximum intensity of the PIC fluorescence signature is referenced "MAX" and the area of the PIC fluorescence signature above the minimum threshold min is referenced as "Aire".
• FIG. 13 shows an example of a histogram of the maximum (MAX) of the fluorescence intensity on the detector 53 at the bottom of the transport channel, that is to say the maximum intensity of the signal of fluorescence detection 53a.
• This histogram shows two different curves slightly shifted in terms of intensity of fluorescence, which can be used to discriminate between two different populations of X and Y spermatozoa.
• discrimination of X and Y spermatozoa based on this detection alone is not optimal, since it strongly depends in particular on the quality of the spermatozoa. semen, tagging protocol and compression fluids used to guide sperm into the transport channel.
• the yield of the detection is low because only a fraction of the spermatozoa can in practice be discriminated with sufficient reliability.
• FIG. 14 shows an exemplary histogram of the durations D of the fluorescence signatures detected laterally (signal 50a) above said predefined threshold (Smin) of fluorescence.
• This figure shows that the populations X and Y have significantly different emission times D and that the duration D of the fluorescence signature, on the lateral detector 50 (signal 50a), is a parameter which can be used to discriminate the X and X populations of spermatozoa, more reliably and more discriminating than the maximum intensity (MAX) of the fluorescence detected at the bottom of the channel (signal 53a).
• MAX maximum intensity
• FIG. 15 shows an example of a histogram of the durations D of the fluorescence signatures detected at the bottom of the channel (signal 53a) above said predefined threshold (Smin). This figure shows that the X and Y populations have substantially identical emission times and that the duration D of the fluorescence signature on the lateral detector 53
• the electronic processing and control means 7 are designed to process in real time the two fluorescence detection time signals 50a and 53a and to deliver a signal 7a for the control of the selection means 6 in function. of these two fluorescence detection time signals 50a and 53a.
• the processing of the fluorescence detection time signal 50a (lateral detection) consists in measuring, for each signaling fluorescence signature PIC in the signal, at least one parameter which is the emission duration D of said fluorescence detected by said first detection means above the predefined minimum threshold (Smin) of fluorescence referred to above (FIG. 12).
• PIC fluorescence signature
• the electronic processing and control means 7 automatically compare this parameter (duration D) with a predefined and preferably parameterizable maximum threshold, which allows discrimination between the spermatozoa X and Y. When this parameter is below this maximum threshold, the electronic processing and control means 7 automatically detect, for example, that it is a sperm Y and thus making it possible to select a type of spermatozoon. sperm.
• the processing of the fluorescence detection signal 53a consists in measuring, for each PIC fluorescence signature, the maximum intensity of the MAX fluorescence and in comparing this maximum intensity MAX with a predefined maximum threshold Imax which is chosen in a manner to eliminate non-compliant fluorescence signatures and in particular, but not exclusively to discriminate in particular glued spermatozoa. Indeed, in the case of spermatozoa bonded together, it has been shown that the maximum intensity (MAX) of the fluorescence was abnormally high.
• the electronic processing and control means 7 When the electronic processing and control means 7 detect a spermatozoon Y (or respectively an X spermatozoon) on the fluorescence detection signal 50 a (lateral detection) and detect at the same time that the maximum intensity of fluorescence MAX on the signal of detection 53a (channel bottom) is below the preset maximum threshold Imax, they trigger for example a control pulse (control signal 7a) to temporarily stop the selection laser 60 so as not to alter the sperm Y (or respectively a spermatozoon X) which has been detected when it passes in the transport channel in front of the selection laser.
• a control pulse control signal 7a
• FIG. 16 shows a particular example of a fluorescence detection signal 50a and 53 comprising three successive fluorescence signatures referenced respectively PIC1, PIC2, PIC3.
• the PIC1 signature detection signal 50a (lateral detection) has a duration D too important to characterize a spermatozoon Y
• the signature PIC2 of the detection signal 53a (bottom channel detection) has a too high intensity (greater than Imax).
• the electronic processing and control means 7 could be designed to perform detection only from the detection signal 50a (lateral detection).
• the transverse section geometry of the transport channel 100 combined with a hydrodynamic focusing of the spermatozoa makes it possible to spatially orient each spermatozoon SP so that its main surface F of greater surface area is substantially parallel to the main plane of symmetry P1 of the transport channel 100.
• the invention is however not limited to the particular geometry of the transport channel 100 illustrated in the accompanying figures, but extends in particular to any device in which the sperm can be transported in a transport channel one behind the other having substantially the same spatial orientation.
• the transport channel could for example have a circular cross section and the device could be equipped, in a manner known per se, with a nozzle making it possible to inject the spermatozoa into said transport channel by conferring substantially the same spatial orientation in the transport channel.
• the selection means 6 previously described may be replaced by sorting means which make it possible to separate the spermatozoa (SP) of a first type (for example spermatozoid type X) from the spermatozoa (SP) of a second type (for example type Y sperm), without necessarily altering one of the two types of spermatozoa.
• SP spermatozoa

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